Preparation of protoplasts is well known, especially in gram-positive microorganisms. The technology for protoplast formation in both gram-positive and gram-negative microorganisms is discussed extensively by Hopwood, "Genetic Studies with Bacteria Protoplasts," Ann. Rev. Microbiol. 1981. 35: 237-72. The preponderance of techniques discussed by Hopwood related to protoplast formation in Streptomyces and Bacillus organisms. The transformation of protoplasts is described at pages 263-268. Protoplast fusion techniques are reviewed by Peberdy, Enzyme Microb-Technol., 1980, vol. 2, pages 23-29. Protoplast fusion techniques for obtaining genetic recombination in Brevibacterium flavum are discussed by Kaneko et al. in Agric. Biol. Chem., 43 (5), 1007-1013 (1979). Protoplast fusion in Corynebacterium organisms is described by Katsumata et al. in published Japanese patent application No. 56-109587 (publication date Aug. 31, 1981). The process described by Katsumata et al. involves the use of penicillin and lysozyme. Canadian Pat. No. 1,105,859 also describes protoplast fusion as applied to Streptomyces.
Preparation of "co-integrate" plasmids for transformation of Streptomyces and Escherichia coli is described in U.S. Pat. Nos. 4,273,875 and 4,332,900. EPO application No. 058,889 (Katsumata and Furuya) describes plasmid cloning vectors for Corynebacterium.
The following microorganisms utilized in the invention are available from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., U.S.A.
A. Corynebacterium glutamicum, NRRL No. B-15340, a species containing the pSR1 plasmid described in Example 1. PA0 B. Bacillus subtilis, NRRL No. B-15305, a species containing the pBD8 plasmid described in Example 2. PA0 C. Bacillus subtilis, NRRL No. B-15306, a species containing the pBD10 plasmid described in Example 2. PA0 D. Bacillus subtilis, NRRL No. B-15341, the species used for transformation in Example 4. PA0 E. Bacillus subtilis, NRRL No. B-15342, a transformed species containing the pHY47 hybrid plasmid described in Example 4. PA0 F. Bacillus subtilis, NRRL No. B-15307, a transformed species containing the pHY416 hybrid plasmid described in Example 4. PA0 G. Corynebacterium glutamicum, NRRL No. B-15302, a transformed species containing the pHY416 plasmid described in Example 6. PA0 H. Corynebacterium glutamicum, NRRL No. B-15303, a transformed species containing the pHY47 plasmid described in Example 6. PA0 I. Corynebacterium glutamicum, NRRL No. B-15304, another transformed (NRRL B-15301) species also containing the pHY416 plasmid described in Example 6. PA0 J. Corynebacterium glutamicum, NRRL No. B-15301, the species used for transformation in Example 7. PA0 A. Corynebacterium glutamicum, ATCC No. 13059, the species used for transformation in Example 5.
Additionally, the invention utilizes the following organisms already deposited with the American Type Culture Collection of Rockville, Md., U.S.A.